Animals
C57BL/6 mice were purchased from SLAC Laboratory Animal (Shanghai, China). All experimental protocols were approved by the Institutional Animal Care and Use Committee at Shanghai Jiaotong University School of Medicine (Protocol A-2019-060). Mice were housed under controlled temperatures (25 °C) in a 12-hour light/dark cycle (light time, 7 AM to 7 PM) with water and ad libitum chow.
AAV plasmid construction and preparation
The Institutional Biosafety Committee of the Shanghai Jiao Tong University School of Medicine approved AAV procedures. AAV serotype 2/2 was used for all AAVs. The AAV construct backbone (AAV2-CAG-EGFP-WPRE-SV40pA) for shRNAs was purchased from Taitool Bioscience Co. (Shanghai, China). Two targeting sequences against Porf2 (shRNA1, GCCCTTGATTCCTCATGAA; shRNA2, GCACGTAGCCCTAGAGATA) were cloned into pAAV plasmids. The AAV titer used for this study was 1.02E + 13 v.g./mL.
Intravitreal injection and optic nerve crush injury
Intravitreal virus injection, ONC injury, axon labeling, and RGC survival analyses were performed as described previously [2, 6, 24]. Briefly, under anesthesia, 2 μL AAV2-shRNA was injected into the left vitreous humor of a mouse using a WPI Nanofil syringe (35-gauge needle). Two weeks later, the left optic nerve was exposed intraorbital and crushed approximately 1 mm behind the optic disc using a pair of Dumont #5 forceps (Fine Science Tools) for 5 s. To label RGC axons in the optic nerve, 1.5 μL Alexa Fluor 555-conjugated CTB (2 mg/ml, Thermo Fisher Scientific) was injected into the left vitreous humor using a WPI Nanofil syringe (35-gauge needle) two days before the mouse was sacrificed by transcardial perfusion under anesthesia. For the post-injury treatment model, all steps were performed in the same way except that intravitreal viral injection was performed one day after the ONC injury.
Retina explant culture
Retinal explant dissection and culturing were performed as described previously [41, 42]. Two weeks after intravitreal injection of AAV2-shPorf-2 or AAV2-shCtrl, mouse eyes were quickly removed using Dumont #5 forceps (Fine Science Tools) and placed into ice-cold Neurobasal-A medium (Invitrogen, 10888022) without calcium and magnesium. Under the anatomic microscope, the dissected eyes were cut open to obtain the retina, which was cut into small pieces approximately 750 μm in diameter with a punch needle. Then, the Neurobasal-A medium was removed from coverslips previously coated with poly-L-lysine and laminin, and the retina explants with an RGC layer were placed face down on the coated side of the glass coverslip. Finally, retinal explants were cultured in Neurobasal-A medium with the following supplements: 2% B27 (Invitrogen, 0080085-SA), 0.5% L-Glutamine (Corning, 25-005-CI), 0.4% methylcellulose (Sigma-Aldrich, M0512), 100 units/mL penicillin–100 μg/mL streptomycin (Invitrogen, 15140122). Note that all explants adhered to the glass coverslip. If an explant became detached, it was removed from the culture. Retinal explants were cultured at 37 °C with 5% carbon dioxide for one week.
Histology and immunostaining of retinal sections, whole-mount retinas, and retinal explants
Histology and immunostaining were conducted as described in our previous study [2]. Briefly, eyes with the attached optic nerve segment, which had been surgically removed from perfused mice, were post-fixed in 4% PFA for two hours. Typically, 20 mm-thick sections were cut for retinas, and 14 mm-thick longitudinal sections were cut for optic nerves. For immunostaining, the sections were blocked in a staining buffer containing 3% bovine serum albumin, 5% normal goat serum, and 0.4% Triton X-100 in phosphate-buffered saline (PBS) for two hours. The sections were incubated with primary antibodies overnight at 4 °C and subsequently incubated with secondary antibodies at room temperature for two hours. For whole-mount retinas, fixed retinas were first radially cut into a petal shape (4 incisions) and blocked with PBS containing 3% bovine serum albumin, 5% normal goat serum, and 0.6% Triton X-100 for two hours at room temperature. Then, retinas were incubated with primary antibodies in a blocking buffer for two days at 4 °C. Next, retinas were incubated with secondary antibodies in a blocking buffer for two hours at room temperature. Images were captured using a confocal laser microscopy (TCS SP8, Leica, Germany).
For the immunostaining of retina explants, we used a previously published protocol [26, 42]. First, retinal explants were fixed in 3% PFA/3% sucrose for 15 min, then blocked with PBS containing 3% bovine serum albumin, 5% normal goat serum, and 0.1% Triton X-100 for 10 min at room temperature. Next, the explants were incubated with a primary antibody (anti-beta III tubulin, Tuj1) for two hours. Finally, the explants were washed three times for 10 min each with PBS and incubated with secondary antibodies and phalloidin (Invitrogen, R415) for one hour.
Primary antibodies used: Guinea pig anti-RBPMS (1:200, MilliporeSigma, ABN1376), Mouse anti-beta III tubulin (Tuj1, 1:300, Abcam, ab78078), Rabbit anti-Porf-2 (1:200, Abcam, ab93780), Rabbit anti-pGSK3β (Ser9) (1:200, Cell Signaling Technology, 5558), Rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (1:200, Cell Signaling Technology, 4858). Secondary antibodies used: Goat anti-Mouse IgG (H + L) Alexa Fluor 488 (1:500, Thermo Fisher Scientific, A11001), Goat anti-Rabbit IgG (H + L) Alexa Fluor 555 (1:500, Thermo Fisher Scientific, A21428), Goat anti-Mouse IgG (H + L) Alexa Fluor 633 (1:500, Thermo Fisher Scientific, A21050), Goat anti-Guinea pig 488 (1:500, Abcam, ab150185), and Goat anti-Guinea pig 647 (1:500, Abcam, ab150187).
Western blot analysis of mTORC1, GSK3β and STAT3 pathway-related proteins
Total protein was extracted from retinal tissues using the RIPA buffer (Epizyme, PC102) supplemented with protease inhibitor and phosphatase inhibitor cocktail. The extracted proteins were then separated by 10% gradient SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Subsequently, the membranes were blocked for 2 h in TBST (150 mM NaCl, 10 mM Tris, 0.1% Tween 20, pH 7.6) containing 10% BSA. Primary antibodies were diluted in blocking buffer and incubated with the membranes overnight at 4 °C. After washing the membranes with TBST three times, they were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Following another round of washing with TBST three times, the blots were exposed to an enhanced chemiluminescence substrate. Quantification of the bands was performed by analyzing the relative density of the exposed film using Image J software. The primary antibodies used for Western blotting were as follows: anti-phospho-STAT3 antibody (1:1000, Cell Signaling Technology, 9145), anti-STAT3 antibody (1:1000, Cell Signaling Technology, 9139), anti-phospho-GSK3β antibody (1:1000, Cell Signaling Technology, 5558), anti-GSK3β antibody (1:1000, Cell Signaling Technology, 12456), anti-phospho-S6 antibody (1:1000, Cell Signaling Technology, 5364 s), anti-S6 antibody (1:1000, Cell Signaling Technology, 2217 s), anti-phospho-4EBP1 antibody (1:1000, Cell Signaling Technology, 2855), anti-4EBP1 antibody (1:1000, Cell Signaling Technology, 9644), and anti-GAPDH antibody (1:1000, Cell Signaling Technology, 51332).
Quantification of fluorescence intensity, RGC survival rate, RGC axon regeneration, retinal explant growth, and axon growth cone formation
Fluorescence images were taken in the same configuration for all retinas, and the confocal settings were kept constant for all scans. Fluorescence intensity was analyzed using ImageJ software. In all sections, RGCs were circled according to their morphology based on RBPMS or Tuj1 staining. To quantify the fluorescence intensity of Porf-2, pS6, or pGSK3β in all RGCs, five mice from each group were used, and at least eight non-adjacent retinal sections acquired with identical imaging settings were analyzed for each mouse.
To quantify RGC survival rate, retinal sections and whole-mounts were immunostained with an anti-RBPMS or anti-Tuj1 antibody. For the retinal sections, at least eight non-adjacent retinal sections were analyzed for each mouse. For retinal whole-mounts, the entire retina was divided into four quadrants centered on the optic papilla, and a consistent number of fields (six to nine) were selected in the center, middle, and periphery of each quadrant. Only RGCs in the ganglion cell layer were counted.
Regenerating RGC axons in injured optic nerves distal to the crush site were quantified as described previously [2, 7]. The number of CTB-labeled axons was estimated by counting the number of CTB-labeled fibers extending different distances from the end of the crush site in four sections per optic nerve. The cross-sectional width of the nerve was measured at the counting point to calculate the number of axons per millimeter of nerve width. The number of axons per millimeter was averaged over all sections. Σad, the total number of axons extending distance d in a nerve with a radius of r, was estimated by summing over all the sections of a thickness t (14 µm): Σad = πr2 x [average axons/mm]/t.
Quantification of retinal explant growth and axon growth cone formation was performed as described previously [26, 42]. Anti-Tuj1 and Phalloidin were used to mark the morphology of axons and the growth cones in retinal explants, respectively. The axon outgrowth from retinal explants and growth cone area were quantified using ImageJ software.
Analysis of RGC transduction rate
Analysis of the RGC transduction rate was conducted based on previous studies [2, 24]. Uninjured left retinas were taken from transcardially perfused mice two weeks after intravitreal AAV2-EGFP vector injection. The retinas were stained with a guinea pig anti-RBPMS antibody (1:200, MilliporeSigma, ABN1376) following the steps described above. For each mouse, the RGC transduction rate was calculated by dividing the total number of EGFP+ and RBPMS+ co-labeled cells by the total number of RBPMS+ cells. Only cells in the GCL were counted.
Drug handling and administration
The Rac1 inhibitor, NSC23766 (Selleckchem, S8031), was administered as described previously [37, 38]. For in vivo studies, 4.0 mg/kg NSC23766 or the vehicle control was administered intraperitoneally once every two days following the AAV2-shPorf-2 injection. For in vitro experiments, 30 μM NSC23766 or the vehicle was added to the retinal explant medium, and the medium was changed once every two days. The detailed culture methods for retinal explants are described above.
Rac1 activation assay and western blot analysis
Rac1 activity was assessed by calculating Rac1-GTP/Total Rac1 using the Rac1 Activation Assay Kit (Cytoskeleton, BK035-S) according to the manufacturer’s instructions. First, total protein was extracted from retinal tissues using the lysis buffer with the protease inhibitor and phosphatase inhibitor. 300–800 µg total protein was incubated with 10 µg PAK-PBD beads to pull down activated Rac1 proteins. After incubating at 4 °C on a rotator or rocker for 1 h, the PAK-PBD beads were pelleted by centrifugation at 5000 × g at 4 °C for 1 min. Next, 90% of the supernatant was removed very carefully, and the PAK-PBD beads were washed three times with a cold wash buffer. Then, 10–20 µl of 2x laemmli sample buffer was added to each tube, and the beads were thoroughly resuspended. Finally, the bead samples were boiled for 2 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB) analyses.
The extracted proteins were separated using SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Then, membranes were blocked for 2 h in TBST (150 mM NaCl, 10 mM Tris, 0.1% Tween 20, and pH 7.6) containing 10% BSA. Primary antibodies were diluted in blocking buffer and incubated with the membrane overnight at 4 °C. After washing with TBST three times, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing with TBST three times, the blots were exposed to an enhanced chemiluminescence substrate. Quantification was performed by analyzing the relative density of the exposed film using Image J. The following primary antibodies were used for WB: anti-Rac1 monoclonal antibody (1:1000, cytoskeleton, Cat # ARC03) and anti-GAPDH (1:1000, arigo, ARG10112).
SD-OCT examination
SD-OCT (Micron IV; Phoenix Research Laboratories, Pleasanton, CA, USA) was used to measure the changes in GCC thickness before and three weeks after ONC injury, as previously described [2, 43]. Briefly, radial volume scans (centered on the optic disc, with a diameter of 1.2 mm) were performed, and each volume consisted of 100 B-scans with 1000 A-scans per B-scan. Four images (scans 1, 26, 51, and 76 at 0°, 45°, 90°, and 135° in en face images) were analyzed using InSight software (version 1.1.5207, Phoenix Research Laboratories) and used for retinal thickness measurements. For each selected image, a vertical caliper was placed on each side of the optic nerve head (ONH) 500 µm away from the center of the ONH. The caliper was used to measure the thickness of the GCC, which consisted of the three innermost retinal layers: the NFL, GCL, and IPL. The GCC thickness of each retina was taken as the average of a total of eight measurements. The image analysis was performed in a blinded manner.
pSTR-ERG examination
pSTR-ERG was performed to assess functional changes in RGCs before and three weeks after ONC injury, as previously reported [44, 45]. Mice were dark-adapted for 12 h and prepared for pSTR-ERG recording under dim red light. After the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg), their pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride. The recording electrode attached to a contact lens was placed on the center of the cornea. A 2.5% hypromellose ophthalmic solution was applied to maintain hydration and conductivity between the cornea and the recording electrodes. The reference electrode was placed in the middle of the lower eyelid, and the ground electrode was placed near the tail of the mouse. Recordings were generated using the Espion Visual Electrophysiology System (Espion E3, Diagnosys, Diagnosys UK Ltd, UK). The ERG was recorded under dark adaptation with increasing stimulus intensity from 3 × 10−5cd.sm−2 to 0.03 cd.sm−2. The response to approximately 30 flashes was recorded at each stimulus intensity, with an interstimulus interval of 2 s, and then averaged to determine the final value of pSTR. The amplitude of pSTR was measured from the baseline to the peak of the first positive wave.
PLR examination
PLR was performed to evaluate the functional responses of the retina and the optic nerve before and eight weeks after ONC injury, as previously reported [46, 47]. Before the experiment, animals were dark-adapted for at least 12 h. Under dim red illumination, each mouse was placed in a head-and-body restrainer device with a metal bar (implanted into the skull two days before the recording) fixed to the device and PLRs were recorded using a pupillometer (A2000; Neuroptics Inc., Irvine, CA, USA) in combination with Bandicam software (Bandicam Company, Seoul, South Korea). After 60 s of dark adaptation, PLR was recorded at a light intensity of 1 W/m2, and the stimulus was presented for 20 sec. Pupil area was then quantified manually using ImageJ software. The percent change in pupil area was calculated by recording the pupil area in the dark, subtracting the pupil area in the light, dividing the difference by the pupil area in the dark, and multiplying by 100. To facilitate a better understanding of the variations in pupil constriction among the different groups, we normalized the results to the pupil constriction in the intact group. This normalization enables a comparison of relative changes in pupil size across the various experimental conditions. The mean percent constriction of each pupil was utilized for statistical analyses.
Statistical analysis
GraphPad Prism software was used for statistical analysis. All statistical details of each experiment are depicted in the figure legends. The data shown in the graphs are presented as the mean ± SEM. Two-tailed unpaired t-tests or Mann–Whitney test were used for comparisons between the two groups. Comparisons between two groups at multiple time points were analyzed via two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparisons test. One-way ANOVA with Bonferroni’s multiple-comparisons test was used to compare multiple groups. A P-value % 0.05 was considered statistically significant.